Aim 2: Determine the sensitivity of OMI to breast cancer subtypes and therapeutic response in vitro and validate OMI measures of response in human breast cancer xenografts
4.3 Materials and Methods
4.3.1 Fluorescence lifetime instrumentation
A custom built, commercial multi-photon fluorescence microscope (Prairie Technologies) was used to acquire fluorescence images. A 40X water-immersion objective (1.15 NA) or a 40X oil-immersion objective (1.3 NA) coupled the excitation and emitted light through an inverted microscope (TiE, Nikon). A titanium:sapphire laser (Coherent Inc.) was tuned to 750 nm for excitation of NADH and 890 nm for FAD excitation. The average laser power was 7.5-7.8 mW for NADH and 8.4-8.6 mW for FAD. A pixel dwell time of 4.8 s was used. A GaAsP PMT
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(H7422P-40, Hamamatsu) detected emitted photons. A 400-480 nm bandpass filter isolated NADH fluorescence. A 500 nm high pass dichroic mirror and a 500-600 nm bandpass filter isolated FAD fluorescence.
Fluorescence lifetime images were acquired using time correlated single photon counting (TCSPC) electronics (SPC-150, Becker and Hickl). TCSPC uses a fast detector PMT to measure the time between a laser pulse and fluorescence event. Each image of 256x256 pixels was acquired using an integration time of 60 seconds. No change in the photon count rate was observed, ensuring that photobleaching did not occur. The instrument response function (measured from the second harmonic generated signal of urea crystals excited at 900 nm) full width at half maximum was measured to be 260 ps. The single-component fluorescence lifetime of a fluorescent bead (Polysciences Inc.) was measured daily. The measured fluorescence lifetime of the bead was 2.1
± 0.08 ns (n = 18), which is consistent with published studies (20, 27).
4.3.2 Cell culture
All cell lines were acquired from the ATCC except the HR6 cell line (28) which was provided by the Arteaga lab. The non-cancerous mammary epithelium cell line, MCF10A, was cultured in MEBM (Lonza) supplemented with cholera toxin, penicillin: streptomycin, bovine pituitary extract, hydrocortisone, insulin, and human epidermal growth factor. All malignant cell lines were grown in DMEM (Invitrogen) with 10% fetal bovine serum and 1% penicillin:
streptomycin. The growth media for the HR6 cell line was further enhanced with 25 g/ml trastuzumab (Vanderbilt Pharmacy). For fluorescence imaging, cells were plated at a density of 106 cells per 35 mm glass-bottom imaging dish (MatTek Corp.) 48 hours before imaging.
The MCF10A cell line was used as a daily fluorescence standard for the redox ratio, and imaged each day measurements were acquired. All other cell lines were imaged on at least two
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different days. A total of 18 different locations were imaged for each cell line (58 for MCF10A cells) from six different dishes (three images were acquired from each dish, see Appendix A Table A.1).
4.3.3 Cyanide experiment
NADH and FAD fluorescence lifetime images of three locations of three dishes were acquired. Media of two of the MCF10A dishes was removed and replaced with cyanide supplemented MCF10A growth media (4 mM NaCN, Sigma). The cells were allowed 5 minutes for the cyanide to react, and post-cyanide NADH and FAD fluorescence images were acquired from three unique locations from each dish.
4.3.4 Trastuzumab perturbation
The effect of HER2 inhibition by trastuzumab was tested in HER2-overexpressing cells.
The cells were plated at a density of 106 cells per imaging dish, 48 hours before imaging. At 24 hours before imaging, the growth media was exchanged for growth media containing 25 g/ml trastuzumab. This dose of trastuzumab, 25 g/ml, was chosen to mimic therapeutic drug dosage in patients (29).
4.3.5 Mouse xenografts
This study was approved by the Vanderbilt University Animal Care and Use Committee and meets the National Institutes of Health guidelines for animal welfare. MDA-MB-361 cells (106), BT474 cells (108), or HR6 cells (108) in 100µl Matrigel were injected in the inguinal mammary fat pads of female athymic nude mice (J:NU; Jackson Laboratories). Tumors were allowed to grow to ~150mm3. Tumor-bearing mice were treated with trastuzumab (Vanderbilt University Medical Center pharmacy) or control human IgG 10 mg/kg twice weekly for two weeks. This dose of trastuzumab was chosen to mimic therapeutic drug dosage in patients (30).
69 4.3.6 OMI xenograft imaging
Isoflurane-anesthetized mice were used for vital imaging, by removing the skin overlying the tumor, overlaying the tumor with a coverslip, and placing the mouse on the microscope stage.
NADH and FAD fluorescence lifetime images of three different tumor locations were acquired each day. After imaging, mice were humanely euthanized while under anesthesia. Each OMI group contained 3 mice, each with 2 tumors for a total n of 6 tumors at each time point.
4.3.7 FDG-PET imaging
The FDG-PET protocol follows published methods (7, 31, 32). The mice were fasted overnight and allowed to acclimate to the PET facility for 1hr on a warm water pad. A single retroorbital injection of ~200 Ci (100l) of [18F]FDG was administered. Following a 40-min distribution period, 20-min static PET scans were collected on a Concorde Microsystems microPET Focus 220 (Siemens) while mice were anesthetized with isoflurane. PET images were reconstructed using the ordered subsets expectation maximization algorithm (33). FDG-uptake values were obtained by isolating the uptake of each tumor volume and correcting for the injected dose. Each FDG-PET group contained 5 mice, each with 2 tumors for a total n of 10 tumors.
4.3.8 Quantification of the optical redox ratio
The optical redox ratio was computed from the NADH and FAD fluorescence lifetime data.
The photons detected at each pixel in an image were integrated over time to compute the sum of photons per pixel. The total number of NADH photons was divided by the total number of FAD photons at each pixel to create a redox ratio image (Matlab, MathWorks). The redox ratio image was thresholded to remove background and nuclear fluorescence and the average redox ratio for the remaining cell cytoplasms was computed. This approach has been confirmed to be consistent with redox ratios obtained with steady-state detection (8, 21).
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4.3.9 Quantification of fluorescence lifetime components
For each image, a threshold was selected to eliminate background and nuclear fluorescence (SPCImage, Becker and Hickl). A binning of nine surrounding pixels was used. Then, the fluorescence lifetime components were computed for each pixel by de-convolving the measured system response and fitting the resulting exponential decay to a two-component model,
C t
I( )1expt/12expt/2 , where I(t) is the fluorescence intensity at time t after the laser excitation pulse, 1 and2 are the fractional contributions of the short and long lifetime components, respectively (i.e. 1 +2 = 1), 1 and2 are the fluorescence lifetimes of the short and long lifetime components, and C accounts for background light. A two-component decay was used to represent the lifetimes of the free and bound configurations of NADH and FAD (20, 23, 24). The average lifetime component values and a mean fluorescence lifetime
(m 11 22) for each image were computed in Matlab.
4.3.10 Statistical analysis
A rank sum test of means was used to test for significant differences due to cyanide. A Bonferroni correction for multiple-comparisons was used on rank sum tests of means of the metabolic values from the panel of cell lines. A rank sum test of means was used to identify significant differences when cell lines were treated with trastuzumab and to find differences in the in vivo xenograft experiments. A student's t-test of means tested for significantly different FDG uptake values between control and trastuzumab-treated xenografts. For all statistical tests, an alpha significance level of 0.05 was used and the test was assumed to be two-way.
Spearman's rank correlation coefficient was used to identify correlations. Both a correlation coefficient (r) and a P-value were computed. An alpha level of less than 0.05 signified
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significance. Scatterplots of the significant correlations confirmed that the correlation was due to data trends and not a single outlier.
4.3.11 Computation of intra- and inter- cellular variation
Inter-cellular variation was visualized by histogram representation of the mean metabolic measure (optical redox ratio, NADH m, or FAD m) for all cells. The histogram was fit to one, two, and three component Gaussian curves to determine the number of modes within the data. The fit with the lowest Akaike information criterion (AIC) was selected to represent the probability density function of the histogram (34). Intra-cellular variation was computed as the average coefficient of variation (standard deviation divided by mean) for each cell and averaged over all cells.
4.3.12 Percentage of mitotic cells
The percentage of proliferating cells was measured by flow cytometry. Cells were plated at a density of 106 cells per 35 mm dish. After 48 hours, the cells were labeled with Phospho- Histone H3 (Ser10) antibody (Cell Signaling Technology) and a secondary antibody, Alexa Fluor 488 goat anti-rabbit IgG (Invitrogen) enabled detection of labeled cells by flow cytometry.
4.3.13 Glycolytic Index
Media glucose and lactate concentrations were measured using standard assay kits (Amplex® Red Glucose/Glucose Oxidase Assay Kit (Invitrogen) and L-Lactate Assay Kit (Eton Bioscience Inc.)). Concentrations of glucose and lactate in the cell growth media were determined at the time of plating (0hr) and at the time of imaging (48hr). The “glycolytic index” was computed as the moles of glucose consumed within 48hr divided by the moles of lactate produced in 48hr.
72 4.3.14 Histological analysis
Tumors were collected and placed in buffered formalin, paraffin embedded, sliced, and stained with H&E. Additional slides were stained for Ki-67 and cleaved caspase-3. Staining protocols were verified in positive control samples. The percentage of positively stained cells was quantified from 5 fields of view from 3 tumors in each group.